Abstract

Numerous data showed that 5-hydroxytryptamine-1A (5-HT1A) receptors couple to Gαo/αi proteins for signal transduction. However, the α subunit isoforms really involved in 5-HT1A receptor coupling in brain remain to be identified. Moreover, regional differences in the functional characteristics of brain 5-HT1A receptors have been evidenced repeatedly. Because such differences could be due to variations in G proteins interacting with the same receptor, relevant approaches were used for identifying α subunits physically coupled to 5-HT1A receptors in different regions of the rat brain. Using immunoaffinity chromatography coupled to Western blot detection, 5-HT1A receptors were found to interact equally with Gαo and Gαi3 in the cerebral cortex, mainly with Gαo and weakly with Gαi3 in the hippocampus and exclusively with Gαi3 in the anterior raphe area. In the hypothalamus, 5-HT1A receptors seemed to be coupled to the latter two G proteins plus Gαi1 and Gαz. Complementary experiments based on an antibody capture technique coupled to both classic radioactivity and scintillation proximity assay detections showed that hippocampal 5-HT1A receptor stimulation induced 5′-O-(3-[35S]thio)triphosphate binding to immunoprecipitates with Gαi3 and Gαo antisera. In the anterior raphe, such 5-HT1A receptor-mediated effect was obtained with Gαi3 antiserum only. These results demonstrated the existence of regional differences in the coupling of 5-HT1A receptors to G proteins in the rat brain. In the anterior raphe, 5-HT1A receptors seem to interact specifically with Gαi3, whereas in the hippocampus, they are mainly coupled to Gαo proteins. Such a disparity in G-protein coupling might explain regional differences in adaptive regulations of brain 5-HT1A receptors.

Because of its involvement in various psychiatric pathologies, such as affective disorders, the 5-HT1A type of serotonin (5-hydroxytryptamine, 5-HT) receptors is a subject of great interest. 5-HT1A receptor stimulation has been shown to activate different signaling pathways sensitive to pertussis toxin through Gαi/Gαo proteins (Raymond et al., 1993). Inhibition of adenylyl cyclase (AC) activity mediated by 5-HT1A receptors has been evidenced in most brain regions as a result of Gαi protein activation (De Vivo and Maayani, 1986; Hamon, 1997). In addition, in the hippocampus, 5-HT1A receptors have been shown to mediate G-protein-gated inwardly rectifying K+ current (GIRK) through a direct interaction of the ionic channel with the βγ subunits of the Gαo isoform (Andrade and Nicoll, 1987; Oleskevich, 1995).

The development of heterologous recombinant systems expressing the 5-HT1A receptor, such as COS-7, HeLa, Chinese hamster ovary, Sf9, GH4C1, LLC-PK1, and NIH-3T3 transfected cells, has provided relevant models to study the receptor coupling with different G-protein subtypes and to identify second messengers. These studies led to the conclusion that 5-HT1A receptors preferentially interact with Gαi3 subunits, followed, in decreasing affinity order, by Gαi2 and less strongly with Gαo, Gαi1, and Gαz proteins (Fargin et al., 1991; Bertin et al., 1992; Liu et al., 1994; Garnovskaya et al., 1997; Newman-Tancredi et al., 2002). In contrast, 5-HT1A receptor coupling to Gαq and Gαs seemed to be weak or absent in such heterologous recombinant systems (Raymond et al., 1993).

The complexity of 5-HT1A receptor coupling, evidenced in recombinant systems, matches the agonist-directed trafficking of receptor signaling theory (Kenakin, 1995). This concept suggests that, depending on the nature of the agonist used, receptors will selectively activate one specific G-protein subtype and downstream transduction pathway. Moreover, this theory also stresses the influence of the “receptor/G-protein ratio” on both the nature of the G protein involved in receptor signaling and the agonist efficacy (Kenakin, 1995; Newman-Tancredi et al., 1997).

In this context, it is well-established that 5-HT1A receptor ligands may act as full agonists in the dorsal raphe nucleus (DRN) but only as partial agonists in the hippocampus (Sprouse and Aghajanian, 1988). Another example of 5-HT1A receptor functional heterogeneity that might also be relevant to this theory concerns the regional differences in 5-HT1A receptor adaptive changes caused by long-term modifications in central 5-HT neurotransmission. Thus, long-term treatment with selective serotonin reuptake inhibitors (SSRIs) and 5-HT transporter (5-HTT) gene disruption are well-known to induce functional desensitization of 5-HT1A auto-receptors in the DRN but no changes in postsynaptic 5-HT1A sites in the hippocampus (Chaput et al., 1986; Le Poul et al., 2000; Mannoury la Cour et al., 2001). In the DRN, this adaptive regulation is associated with a decrease in 5-HT1A receptor-mediated [35S]GTPγS binding, suggesting an alteration of the receptor/G-protein coupling in both SSRI-treated rodents (Hensler, 2002) and 5-HTT knockout mice (Fabre et al., 2000). In the hypothalamus, the 5-HT1A receptor desensitization that occurs in these two models was reported to be associated with down-regulation of Gαo, Gαi1, Gαi2, Gαi3, and Gαz proteins (Li et al., 1997; Raap et al., 1999). Taken together, these data support the idea that such regional differences in the functional and adaptive properties of brain 5-HT1A receptors are probably underlain by variations in receptor coupling to G proteins from one area to another.

Materials and Methods

Experiments were performed using adult male Sprague-Dawley rats (250-400 g body weight; Centre d'Elevage René Janvier, Le Genest-Saint-Isle, France). Animals were maintained under standard laboratory conditions (22 ± 1°C, 60% relative humidity, 12-h light/dark cycle, food and water ad libitum) for 5 to 10 days before the beginning of experiments. All the procedures involving animals and their care were conducted in conformity with the institutional guide-lines that are in compliance with national and international laws and policies (Council directive 87-848, 1987 October 19, Ministère de l'Agriculture et de la Forêt, Service Vétérinaire de la Santé etdela Protection Animale, permissions number 75-116 to M.H. and 75-977 to L.L).

Preparation of Membranes. Rats were killed by decapitation. Their brains were quickly removed, and the cerebral cortex, striatum, hippocampus, cerebellum, hypothalamus, and anterior raphe area were dissected in cold (0-4°C) and stored at -80°C before use. Frozen tissues were homogenized in 10 volumes (v/w) of ice-cold 0.05 M Tris-HCl containing 0.01 mM phenylmethylsulfonyl fluoride and 0.01 mM aprotinin, pH 7.4, with a Polytron tissue disrupter (PT OD; Kinematica, Basel, Switzerland). Homogenates were centrifuged at 40,000g for 20 min at 4°C. The pellets were washed twice by resuspension in 40 volumes of the same ice-cold buffer, followed by centrifugation and homogenization in the same volume of buffer. The resulting membrane suspension was incubated for 10 min at 37°C to allow the dissociation of membrane-bound endogenous 5-HT and then centrifuged and washed three more times as described above. The final pellet was gently homogenized in an appropriate volume of 0.05 M Tris-HCl, pH 7.4, to obtain the membrane suspension (∼20 mg of membrane proteins per milliliter) to be stored at -80°C until use.

Solubilization Procedure. Thawed membrane suspension was mixed with 0.1 volume (v/v) of 0.1 M [0.6% (w/v)] CHAPS in 0.05 M Tris-HCl, pH 7.4, then briefly sonicated (20 W/5 s) and left for 60 min at 4°C (El Mestikawy et al., 1988). The mixture was then centrifuged at 100,000g for 30 min at 4°C. The clear supernatant was collected and filtered through a 0.22-μm Millex GV filter (Millipore Corporation, Billerica, MA) before its use as the source of solubilized 5-HT1A binding sites in subsequent assays. The protein concentration in the final soluble extract was ∼6 mg/ml.

Immunoaffinity Chromatography, Elution, and Concentration Procedures. The anti-rat 5-HT1A receptor polyclonal antibody was purified (El Mestikawy et al., 1990; Riad et al., 1991) and coupled to an Affigel-10 column (2 cm high, 1.5 cm in diameter) as recommended by the manufacturer (Bio-Rad, Hercules, CA). The filtered supernatant (∼4 ml) from CHAPS-treated membranes was poured into the affinity column equilibrated with 0.05 M Tris-HCl, pH 7.4. After an overnight incubation at 4°C, the supernatant was removed, and the column was washed four times with 40 ml of 0.05 M Tris-HCl, pH 7.4, containing 0.1 M CHAPS and then with the same volume of the same buffer containing 0.01 M CHAPS.

Elution was performed in two steps. First, the Gα protein subunits specifically coupled to 5-HT1A receptors were eluted within 15 min, at room temperature, with 5 ml of 0.05 M Tris-HCl, pH 7.4, supplemented with 1 mM 5-HT and 1 mM GTP. The column was then washed with 4 × 20 ml of 0.05 M Tris-HCl buffer, and the second elution was made at 4°C with 5 ml of 0.01 M glycine-HCl, pH 2, containing 0.01 M CHAPS to collect 5-HT1A receptors adsorbed onto the column. Eluate was immediately neutralized with 1 M Tris-HCl, pH 7.4, and dialyzed against 2 liters of 0.05 M Tris-HCl, pH 7.4, containing 0.1% SDS, using a MicroProDiCon apparatus (model MPDC-310; Bio Molecular Dynamics, Beaverton, OR). After 3 days at 4°C, the neutralized eluate was concentrated to a final volume of 300 μl. The same dialysis-concentration procedure was applied to the first eluted fraction (5 ml) containing Gα proteins.

Immunoblot Analysis of Eluted Gα Proteins and 5-HT1A Receptors. The solubilized proteins in concentrated dialysates were analyzed by SDS-polyacrylamide gel electrophoresis using 0.5-mm thick 10% acrylamide/bisacrylamide [29:1 (w/w)] gels with 0.1% SDS and 0.375 M Tris, pH 8.8. After migration, the proteins were electrophoretically transferred for 45 min to a nitrocellulose membrane (Hybond ECL; GE Healthcare, Little Chalfont, Buckinghamshire, UK) that was then incubated, at room temperature, in PBS/0.1% Tween (v/v) containing 5% nonfat dry milk for 1 h. The membrane was subsequently incubated overnight with rabbit polyclonal antibodies directed against either Gαo, Gαi1, Gαi2, Gαi3, Gαz, or Gαs (1:200 dilution) at 4°C. Analysis of the specificity of these anti-Gα antibodies using recombinant Gα subunits showed that no cross-reactivity occurred at the dilution used in our experiments except for anti-Gαi1 and anti-Gαi3, which slightly cross-reacted with Gαi3 and Gαi1 proteins, respectively. The secondary antibody (goat anti-rabbit IgG coupled to horseradish peroxidase conjugate; Sigma, St. Louis, MO) (1:16,000 dilution) was applied to the membrane for 60 min at room temperature. The blot was washed five times with PBS containing 0.1% Tween and once with PBS alone. After a 5-min incubation in ECL Plus chemiluminescence substrate solution (GE Health-care), the membrane was exposed to autoradiography Hyperfilm MP (GE Healthcare) for ∼15 s or analyzed using filmless autoradio-graphic analysis (FLA2000; Fuji, Tokyo, Japan).

[35S]GTPγS Binding and Gα Subunit-Specific Immunoprecipitation. The binding of [35S]GTPγS to specific Gα proteins upon activation of 5-HT1A receptors was measured using a method adapted from Selkirk et al. (2001). A first series of experiments allowed the determination of the optimal assay conditions leading to the highest ratio of 5-HT1A-enhanced over basal [35S]GTPγS binding when starting with hippocampal membranes. On this basis, brain membranes (0.2-1.0 mg/ml) were incubated for 30 min in assay buffer (67 mM Tris-HCl, 4 mM MgCl2, 160 mM NaCl, and 0.267 mM EGTA, pH 7.4) containing GDP (1.2 mM), [35S]GTPγS (0.4 nM), with or without 5-carboxamidotryptamine (5-CT, 10 μM) and WAY 100635 (10 μM, for the determination of nonspecific [35S]GTPγS binding; Fabre et al., 2000) (final volume, 800 μl) in a shaking water bath at 37°C. Incubation was ended by the addition of 500 μl of ice-cold assay buffer and transfer to ice. Membranes were separated from the reaction mix by centrifugation at 20,000g for 6 min, and the supernatant was discarded. Membrane pellets were then solubilized with 500 μl of a solubilization buffer [100 mM Tris-HCl, 1 mM EDTA, 20 mM NaCl, and 0.62% (v/v) Nonidet P-40, pH 7.4] for 1 h at 4°C. Samples were centrifuged (20,000g, 4°C), and 400 μl of the supernatant was incubated with anti-Gα protein antiserum (1/100) during 90 min at 4°C. Protein A-Sepharose beads [70 μl, 50% (w/v)] were added, and samples were rotated for a further 90 min at 4°C. After centrifugation (20,000g, 4°C), the supernatant was removed by aspiration, and the beads were washed three times with 500 μl of solubilization buffer and then resuspended in the same solubilization buffer (500 μl) and filtered through Whatman GF/B filters. After three washes with ice-cold 67 mM Tris-HCl, pH 7.4, each filter was immersed in 5 ml of scintillation fluid, and the entrapped radioactivity was counted. Data are expressed as mean ± S.E.M. of at least three independent experiments.

Scintillation Proximity Assays. SPAs were used to further determine G-protein subtypes specifically activated by 5-HT1A receptor stimulation. The procedure described by Cussac et al. (2002) was adapted so as to be used after [35S]GTPγS binding and solubilization steps (see above). After solubilization, samples (200 μl) were transferred into a 96-well Opti plate (PerkinElmer Life and Analytical Sciences, Boston, MA) and incubated with 2 μl of specific anti-Gα polyclonal antibody (1/100) during 1 h at room temperature. SPA beads coated with secondary anti-rabbit antibodies (GE Healthcare) were then added in a volume of 50 μl/well. After overnight incubation under gentle agitation, the plates were centrifuged (10 min at 1300g), and radioactivity entrapped on beads was measured using a TopCount microplate scintillation counter (PerkinElmer Life Sciences). Data are expressed as mean ± S.E.M. of four independent experiments.

Results

Regional Distribution of Gαo, Gαi1, Gαi2, Gαi3, Gαz, and Gαs Subunits. The presence of Gα-protein subtypes in membranes from the different rat brain regions of interest was investigated using an immunoblotting technique. Rabbit polyclonal antiserums used in these experiments were raised against peptide sequence in highly divergent domains of Gα subunits from rat or human. As shown in Fig. 1, Gαo, Gαi1, Gαi2, Gαi3, Gαz, and Gαs proteins were present in all brain structures studied (hippocampus, anterior raphe area, cortex, and hypothalamus), including striatum and cerebellum, in which 5-HT1A receptors are not detected (Riad et al., 1991).

Immunoblotting with anti-Gαo (Fig. 1A), anti-Gαs (Fig. 1B), and anti-Gαi3 (Fig. 1F) yielded only one band at 45, 47.5, and 40 kDa, respectively. These molecular masses matched those reported in the literature for the three Gα protein subtypes (Schandar et al., 1998). In contrast, a doublet was found with anti-Gαz (Fig. 1C), anti-Gαi1 (Fig. 1D), and anti-Gαi2 (Fig. 1E), the second minor band probably corresponding to a nonspecific signal (Allouche et al., 1999).

Evidence for 5-HT1A Receptor Retention on the Immunoaffinity Column. To validate its capacity to bind 5-HT1A receptors, the immunoaffinity column was loaded with hippocampal CHAPS-solubilized extracts, and glycine-HCl/CHAPS, pH 2, eluates were analyzed by immunoblotting with anti-5-HT1A receptor antibodies. As shown in Fig. 2, these antibodies labeled a single diffuse band at ∼63 kDa in crude hippocampal extracts (Hip), corresponding to the molecular mass of native N-glycosylated 5-HT1A receptor (Emerit et al., 1987; El Mestikawy et al., 1989; Riad et al., 1991). The same heavily labeled band was found in the glycine-HCl fraction eluted from the immunoaffinity column (Fig. 2).

Regional Differences in Gα-Proteins Specifically Coupled to 5-HT1A Receptors. Previous studies have established that neither the solubilization procedure nor antibody binding onto the receptor alters the coupling of 5-HT1A receptor to G protein (El Mestikawy et al., 1988; Emerit et al., 1990). 5-HT1A receptor-G-protein complexes were selectively adsorbed onto the immunoaffinity column, and G proteins were dissociated from these complexes by elution with a mix of 5-HT (1 mM) and GTP (1 mM). Immunoblotting with specific anti-Gα subunit antibodies revealed the presence of Gαo and Gαi3, but not Gαi1, in eluates from immunoaffinity column loaded with cortical 5-HT1A-G protein complexes (Fig. 3A). Similar data were obtained with hippocampal 5-HT1A receptor-G protein complexes (Fig. 3B). However, for the hippocampus, Gαo was more intensely labeled than Gαi3. For the anterior raphe area, only Gαi3 could be detected in eluates from immunoaffinity column loaded with 5-HT1A receptor-G-protein complexes from this region (Fig. 3C). Concerning hypothalamic 5-HT1A receptor-G protein complexes, in addition to Gαo and Gαi3 subunits, we also identified Gαi1 and Gαz in corresponding immunoaffinity column eluates (Fig. 3D).

Immunoblots of Gα subunits in membranes from various rat brain regions. Membrane-bound proteins were solubilized by CHAPS, separated by SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes as described under Materials and Methods. The latter membranes were incubated overnight with 1:200 dilution of anti-sera against Gαo (A), Gαs (B), Gαz (C), Gαi1 (D), Gαi2 (E), and Gαi3 (F). Molecular mass markers, in kilodaltons, are indicated on the left. The first band (from left to right) corresponds to the recombinant protein and the others to various brain structures: Cx, cerebral cortex; Hip, hippocampus; Ra, anterior raphe area; Hy, hypothalamus; Cb, cerebellum; St, striatum.

Immunoaffinity chromatography of hippocampal 5-HT1A receptor. Rat hippocampal 5-HT1A receptor was solubilized and fixed on Affigel-10 immunoaffinity column as described under Materials and Methods. Elution with glycine-HCl (pH 2) allowed the desorption of 5-HT1A receptors which could be visualized on immunoblots with specific polyclonal antibody (left lane, Western blot with 5 μl of glycine-HCl eluate). The right lane (Hip, hippocampus) corresponds to 5-HT1A receptor immunolabeling in soluble extract of rat hippocampal membranes (before loading onto the immunoaffinity column). Molecular mass markers are indicated on the left.

In contrast, immunoblotting analyses of immunoaffinity column eluates yielded no labeling with anti-Gαi2, anti-Gαz, and anti-Gαs in case of 5-HT1A receptor-G protein complexes solubilized from cortical (Fig. 3A), hippocampal (Fig. 3B), and anterior raphe (Fig. 3C) membranes. No immunolabeling with anti-Gαs and anti-Gαi2 was also noted with receptor complexes solubilized from the hypothalamus (Fig. 3D).

As expected from the absence of 5-HT1A receptors in the striatum and the cerebellum (Hamon, 1997), no Gα proteins could be detected in eluates from immunoaffinity columns loaded with soluble membrane extracts from these regions (Fig. 3, A-D).

5-HT1A Receptor Agonist-Induced [35S]GTPγS Binding onto Soluble Extracts from Rat Brain Membranes. Under optimal conditions determined from a preliminary series of experiments (see Materials and Methods), [35S]GTPγS binding induced by 10 μM 5-CT reached ∼60% over basal with hippocampal membranes (Fig. 4, A and B). The percentage of increase produced by 5-CT was less with membranes from the cerebral cortex and the anterior raphe area (Fig. 4A), in line with the lower density of 5-HT1A receptors in these two regions compared with the hippocampus (Fabre et al., 2000).

Immunoprecipitation of Gα Proteins Labeled with [35S]GTPγS in Soluble Extracts from 5-CT-Stimulated Hippocampal Membranes. In a first series of experiments, protein A-Sepharose beads were used to bind immunoprecipitates with various anti-Gα antibodies of [35S]GTPγS-labeled 5-HT1A receptor-G protein complexes solubilized from hippocampal membranes after incubation with or without 5-CT (10 μM). As shown in Fig. 5, A and B, 5-CT-induced [35S]GTPγS labeling of immunoprecipitates was obtained with anti-Gαo- and anti-Gαi3 antibodies. This effect was mediated by 5-HT1A receptor activation because it was completely inhibited in the presence of WAY 100635. In contrast, immunoprecipitation with anti-Gαq, anti-Gαz, and anti-Gαs yielded no 5-CT-induced increase in [35S]GTPγS labeling of immunoprecipitates adsorbed onto protein A-Sepharose beads (Fig. 5C).

Immunoblots of different Gα subunits coupled to 5-HT1A receptors in various brain regions. Membrane-bound proteins were solubilized by CHAPS, and 5-HT1A receptor/G protein complexes were adsorbed onto Affigel-10 immunoaffinity column as described under Materials and Methods. G proteins were eluted by a 5-HT-GTP mix and identified by immunoblotting with specific anti-Gα antibodies (dilution, 1/200). The rp bands correspond to pure recombinant Gα subunits run in parallel with Affigel-10 immunoaffinity column eluates. Note the presence of Gαo and Gαi3 (equally) in the cerebral cortex (A, Cx), of Gαo (mainly) and Gαi3 (trace) in the hippocampus (B, Hip), of Gαi3 (exclusively) in the anterior raphe area (C, Ra), and of Gαo, Gαi1, Gαi3, and Gαz in the hypothalamus (D, Hy). In contrast, no Gα protein immunolabeling is detected with eluates from striatum (St) or cerebellum (Cb) extracts.

SPA Determination of Gα Proteins Labeled by [35S]GTPγS in Soluble Extracts from 5-CT-Stimulated Hippocampal and Raphe Membranes. Additional experiments were performed using an SPA technology with a protocol derived from that used with protein A-Sepharose beads and adapted to 96-well microplates. The high sensitivity of the detection by SPA led us to perform experiments with membranes from the anterior raphe area in addition to hippocampal membranes. A shown in Fig. 6A, 5-CT induced a robust increase in [35S]GTPγS binding to both Gαo- and Gαi3-immunoprecipitated samples, corroborating the results obtained with protein A-Sepharose beads. By contrast, no effect of 5-CT could be detected in assays with anti-Gαs antibodies.

In the anterior raphe area, a significant increase in [35S]GTPγS binding under 5-CT-stimulated compared with basal conditions was measured when immunoprecipitation was made with anti-Gαi3 antibodies (Fig. 6B). In contrast, no 5-CT-induced effect could be detected with anti-Gαo antibodies.

As expected from its mediation through 5-HT1A receptors, the 5-CT-induced increase in [35S]GTPγS labeling of immunoprecipitates with anti-Gαi3 and anti Gαo antibodies was not observed with membranes that had been incubated with both 5-CT and WAY 100635 (Fig. 6, A and B).

Discussion

Using appropriate immunopurification strategies, we herein report the first direct identification of G proteins coupled to native 5-HT1A receptors in the rat brain. Our data were obtained using two complementary approaches that have been used already for either identifying the coupling between a receptor and a G protein or estimating the efficacy of this coupling (Shreeve, 2002). The immunoaffinity copurification technique relies on the high specificity of the anti-serum immobilized on the immunoaffinity column. Previous characterizations have shown that our antiserum selectively immunoprecipitates 5-HT1A but not other 5-HT1 receptors in solubilized hippocampal membranes and that the receptor/G-protein coupling is preserved in the immune complex (El Mestikawy et al., 1990; Riad et al., 1991). Solubilization of tissue extracts was another critical step, because the detergent could induce the dissociation of the 5-HT1A receptor/G-protein complexes. However, we found previously that CHAPS enables the solubilization of functional 5-HT1A receptors physically coupled to G proteins (El Mestikawy et al., 1988; Emerit et al., 1990).

5-CT-stimulated [35S]GTPγS binding onto Gαo and Gαi3 but not Gαq, Gαz, and Gαs subunits in soluble extracts from rat hippocampal membranes: antibody capture assays. [35S]GTPγS binding onto Gαo (A) and Gαi3 (B) subunits from 5-HT1A receptor/G protein complexes solubilized from hippocampal membranes after incubation with 10 μM 5-CT and/or 10 μM WAY 100635. 35S-labeled Gα subunits were immunoprecipitated and then bound onto protein A-Sepharose beads as described under Materials and Methods. In contrast to anti-Gαo and anti-Gαi3 antibodies, anti-Gαq, -Gαz, and -Gαs antibodies (C) did not yield any 5-CT-stimulated 35S-labeling. Results are expressed as the percentage of basal labeling with [35S]GTPγS (100% = 2424 ± 29 and 1410 ± 43 dpm with anti-Gαo and anti-Gαi3 antibodies, respectively) in the absence of 5-CT and WAY 100635. Each bar is the mean ± S.E.M. of triplicate determinations in eight independent experiments. ***, p < 0.001 (paired Student's t test), ns, not significant.

For our immunopurification protocols, we also used immunoprecipitation procedures whose reliability depends on the specificity of anti-Gα antiserums. This critical point has been evaluated using recombinant Gα proteins. No cross-reactivity was observed with the used antibodies, except for those directed against Gαi1 and Gαi3 subunits. Because immunoaffinity chromatography experiments showed that 5-HT1A receptors did not couple with Gαi1 in the hippocampus and the anterior raphe area, it is probable that the radioactivity measured in immunoprecipitates with anti-Gαi3 antibodies resulted from the precipitation of Gαi3- and not Gαi1-[35S]GTPγS-labeled complexes. Concerning the SPA approach, the selectivity of the detection was improved by using secondary antibodies coated on beads that recognize the primary antiserum more specifically than the protein A-Sepharose does. The resulting reduction of the background noise makes this technique more sensitive and enabled the detection of low-intensity signals such as those obtained with raphe membranes (Fig. 6B).

One of the most important observations of our study is that the G-protein coupling of 5-HT1A receptors exhibited clear-cut regional differences in the rat brain. However, all of the G proteins that interact with 5-HT1A receptors in the cortex, hippocampus, hypothalamus, and anterior raphe area belong to the Gi/Go family (Gαo, Gαi1, Gαi3, and Gαz). All of them have already been identified using heterologous coexpression of 5-HT1A receptors with various Gα subunits in recombinant systems (Bertin et al., 1992; Raymond et al., 1993; Garnovskaya et al., 1997).

In vitro data have suggested that 5-HT1A receptor/Gαi2 coupling results in both AC inhibition and increase of intracellular Ca2+ concentration (Raymond et al., 1993; Albert et al., 1996). Despite these data, no band corresponding to Gαi2 was identified in any tested fractions, indicating that, in the rat brain, native 5-HT1A receptors do not activate this G-protein subtype.

Although no direct interaction between Gαs protein and 5-HT1A receptor has been observed in transfected Chinese hamster ovary cells (Raymond et al., 1993), mutations in the third intracellular loop of the 5-HT1A receptor have been shown to induce a weak Gαs coupling (Malmberg and Strange, 2000). Furthermore, both microdialysis studies and biochemical experiments performed in rat hippocampus demonstrated an increased cAMP formation in response to 5-HT1A receptor agonists, such as 8-OH-DPAT and 5-CT, suggesting a positive AC coupling of 5-HT1A receptors through Gαs stimulation (Shenker et al., 1987; Cadogan et al., 1994). However, it has to be stressed that the agonists used in these studies can stimulate other 5-HT receptors in addition to the 5-HT1A type, notably 5-HT7 receptors, which are known to activate AC via Gαs proteins (Hamon, 1997). In fact, our data clearly showed that 5-HT1A receptors interact with Gαs in neither the hippocampus nor any other brain structures examined. In fact, Albert et al. (1999) found that the 5-HT1A receptor-stimulated production of cAMP in HEK 293 cells requires the coexpression of AC type II, which constitutive activation involves βγ subunits probably originating from Gαi2 proteins. Taken together, these data suggest that a positive coupling between 5-HT1A receptors and Gαs might occur only under specific conditions that require both specific cellular milieu and particular AC subtypes that are not found in rat brain extracts.

In agreement with previous data in recombinant systems (Bertin et al., 1992; Raymond et al., 1993; Garnovskaya et al., 1997), clear-cut interaction of native 5-HT1A receptors with Gαi3 subunit was demonstrated in the cortex, the hypothalamus, the anterior raphe region, and the hippocampus of the rat brain. However, in the hippocampus, immunoaffinity chromatography experiments indicated that 5-HT1A receptors coupled mainly with Gαo and, to a lower extent, with Gαi3. On the other hand, immunoprecipitation experiments evidenced that 5-CT similarly increased [35S]GTPγS binding onto both G-protein subtypes, suggesting that 5-HT1A receptors could activate Gαo and Gαi3 with the same efficacy. Such discrepancies might be related to immunoaffinity chromatography conditions, in which only one active state of 5-HT1A receptor could be present and interact essentially with the Gαo subunit. In contrast, in immunoprecipitation experiments, the high-efficacy agonist 5-CT might stimulate two different active states of the receptor coupled to either Gαo or Gαi3 subunit (Kenakin, 1995). On the other hand, Gαo and Gαi3 subunits may display a difference in the rate of GDP dissociation, as already shown in the case of Gαo and Gαi1 (Remmers et al., 1999). Therefore, the Gαi3-GTP complex would be less stable and Gαi3 intrinsic GTPase activity higher than that of Gαo subunit. Such characteristics would explain the lower signal obtained with Gαi3 in immunoaffinity chromatography experiments. Finally, this coupling could also involve specific regulators of G-protein signaling (RGSs). Indeed, differences have been reported between RGS regulating Gαo-versus Gαi-GTPase activity (Lan et al., 2000).

The coupling of native 5-HT1A receptors to Gαo in the hippocampus corroborates previous results from electrophysiological experiments. Relevant studies demonstrated that 5-HT1A receptor stimulation opens a GIRK conductance through the activation of Gαo1-protein subtype in hippocampal granule cells (Oleskevich, 1995). It is interesting to note that more recent data from knockout mice evidenced that, in the hippocampus, Gαo is the predominant G protein used for coupling both GABAB and adenosine receptors to K+ channels (Greif et al., 2000). This conclusion can probably be extended to 5-HT1A receptors, because the latter have been shown to share the same pool of G proteins with GABAB and adenosine receptors in the hippocampus (Mannoury la Cour et al., 2004). In contrast, no interaction with Gαo has been detected in the anterior raphe area in which we found that 5-HT1A receptors physically coupled to Gαi3 only.

This disparity between the hippocampus and the anterior raphe area is particularly relevant regarding the differential regulation of 5-HT1A receptors. Long-term inactivation of 5-HT reuptake by SSRI treatment in rats and 5-HTT gene disruption in 5-HTT-/- mice induce a functional desensitization of 5-HT1A autoreceptors within the DRN but no adaptive changes of 5-HT1A heteroreceptors in the hippocampus (Le Poul et al., 2000; Mannoury la Cour et al., 2001). Transductional modifications are probably at the origin of such regional differences in adaptive regulation of 5-HT1A receptors. Indeed the intronless structure of the 5-HT1A receptor gene is incompatible with the possible existence of several forms of the receptor protein. This desensitization seemed to be associated with a decrease in 5-CT-stimulated [35S]GTPγS binding only in the DRN, suggesting an alteration of receptor/G-protein coupling in this region (Fabre et al., 2000; Hensler, 2002). Our results suggest that desensitization could implicate an alteration of the coupling of 5-HT1A receptors with Gαi3 but not Gαo subunits. It is interesting that a recent in vivo study indicated that overexpression of RGS4 within the DRN attenuated specific Gαi-mediated 5-HT1A receptor signaling, leading to a decrease in 5-HT1A autoreceptor functional response (Beyer et al., 2004). In contrast, such a mechanism would not exist in the hippocampus in which 5-HT1A receptors mediate K+ channel opening essentially through Gαo subunits (Oleskevich, 1995).

Therefore, in regions in which 5-HT1A receptors are coupled with several G proteins, adaptive compensatory changes might occur to preserve the functional characteristics of the receptors. This might take place in the hippocampus (Greif et al., 2000) and in the cerebral cortex, in which the 5-HT1A receptor/G-protein coupling is unaffected by long-term treatment with fluoxetine (Hensler, 2002). In the hypothalamus, 5-HT1A receptors seemed to be coupled to four different Gα subunits, Gαo, Gαi1, Gαi3, and Gαz. In line with our data, a recent study demonstrated the existence of 5-HT1A receptor-Gαz interaction in the hypothalamic paraventricular nucleus using Gαz antisense oligodeoxynucleotides (Serres et al., 2000). It is interesting that hypothalamic 5-HT1A receptors have also been shown to be functionally desensitized after long-term SSRI administration. This adaptive change was reported to be associated with a reduction in the levels of Gαo, Gαi1, and Gαi3 proteins (Li et al., 1997) and a decrease in membrane-bound Gαz protein (Raap et al., 1999). Such a down-regulation of all Gα proteins coupled to hypothalamic 5-HT1A receptors (i.e., the absence of any opposite compensatory changes among these proteins) probably accounts for 5-HT1A receptor desensitization in this particular region.

In conclusion, our data demonstrate that, in the rat brain, regional differences exist regarding the Gα protein subtypes that interact with native 5-HT1A receptors. These differences are particularly striking in the anterior raphe area versus the hippocampus, in which differential adaptive changes in 5-HT1A receptors have been reported repeatedly after long-term blockade of 5-HT reuptake. Determinations of 1) Gαi3 and Gαo mRNA and protein levels, 2) associated Gβ subunits, and 3) the stoichiometry between Gαi3/Gαo and Gβ subunits, specifically in cells expressing 5-HT1A receptors, will ultimately provide key data concerning the regional differences in 5-HT1A receptor signaling and regulation. In addition, deciphering the mechanisms through which differential coupling occurs is definitively the further goal to be achieved. In particular it will be necessary to identify the different partner proteins that interact with these particular G proteins in the transduction cascade downstream of 5-HT1A receptor stimulation. The probable presence of several RGS proteins in 5-HT1A receptor-expressing cells, the heterogeneity of G protein-coupled receptor kinases, and possible regional differences in 5-HT1A receptor-dependent effectors (such as GIRK channels) has also to be considered in studies aimed at explaining the functional and regulatory heterogeneity of brain 5-HT1A receptors.

Acknowledgments

We are grateful to Wyeth-Ayerst for the generous gift of WAY 100635.

Footnotes

This research was supported by grants from the Institut National de la Santé et de la Recherche Médicale (INSERM), Université Pierre et Marie Curie, Bristol-Myers Squibb Foundation (Unrestricted Biomedical Research Grant Program), and the European Community NewMood Program (LSHM-CT-2003-503474). C.M.L.C. was the recipient of fellowships from the Fondation pour la Recherche Médicale and INSERM (poste d'accueil) during performance of this work.

Li Q, Muma NA, Battaglia G, and Van de Kar LD (1997) A desensitization of hypothalamic 5-HT1A receptors by repeated injections of paroxetine: reduction in the levels of Gi and Go proteins and neuroendocrine responses, but not in the density of 5-HT1A receptors. J Pharmacol Exp Ther282:1581-1590.

Malmberg A and Strange PG (2000) Site-directed mutations in the third intracellular loop of the serotonin 5-HT1A receptor alter G protein coupling from Gi to Gs in a ligand-dependent manner. J Neurochem75:1283-1293.